Abstract Their location and highly aggressive nature renders glioblastoma (GBM) among the most deadly and devastating of human malignancies. Despite extensive treatment involving surgery and adjuvant chemo-radiotherapy, the prognosis is still dismal and novel treatment strategies are urgently needed. Of all existing adjuvant therapies, radiotherapy contributes the most to extending the median overall survival. Increasing the efficacy of existing radiotherapeutic regimens is therefore a logical avenue to improve the survival of GBM patients. We have developed a novel radiosensitization strategy called ‘induction of mitotic enrichment’. It has long been known that the radiosensitivity of a cell depends on the phase of the cell cycle and that especially mitotic cells are especially vulnerable. Enriching the tumor for mitotic cells by arresting them during division prior to each radiotherapy fraction should therefore render the tumor population more sensitive to irradiation. Ideally, induction of mitotic enrichment should be reversible and non-cytotoxic to prevent healthy tissue toxicity and be compatible with clinically applied hyperfractionated radiotherapy regimens. We have now identified an orally available targeted tubulin polymerization inhibitor that can achieve repeated and reversible mitotic enrichment for up to 10 hours prior to radiotherapy, without causing cytotoxicity in vitro or healthy tissue toxicity in vivo. Most importantly, this tubulin inhibitor efficiently radiosensitizes a range of preclinical GBM models in vitro and in vivo, including GSC models, and significantly improves survival, but only in a mitotic enrichment setup when given 6-8 hours prior to radiotherapy to allow accumulation in mitosis. We are currently expanding our preclinical development of mitotic enrichment as a radiosensitization strategy to other mitotic targets and different intra- and extracranial cancer models representing several diseases for which radiotherapy is a mainstay treatment. In parallel, we are preparing a phase 0 trial to demonstrate induction of mitotic enrichment in human GBM.